Lactic Acid Bacteria

ABSTRACT

Lactobacillus delbrueckii  having antifungal activity is provided as novel lactic acid bacteria having antifungal activity. The  Lactobacillus delbrueckii  is  Lactobacillus delbrueckii  ANTI MUFFA (FERM P-19705). The  Lactobacillus delbrueckii  has antifungal activity against mold of genus  Penicillium  and is excellent in probiotic activity even when used solo.

TECHNICAL FIELD

This invention relates to novel lactic acid bacteria that haveantifungal activity and that are excellent in probiotic activity.

BACKGROUND ART

Lactic acid bacteria are known to produce a variety of antimicrobialsubstances (antibacterial substances) such as a lactic acid, an organicacid, a volatile fatty acid, a hydrogen peroxide, a benzoic acid, abacteriocin (niacin, colicin or the like) and so on. The lactic acidbacteria are used not only for a dairy starter but also for preservationof foods (biopreservation) by use of an antibacterial activity. However,not only bacteria propagate themselves as pathogenic bacteria orputrefactive bacteria, but also mold propagates itself during thepreservation of the foods. Thus, most of the lactic acid bacteria werenot effective against propagation of the mold. Particularly, the moldtends to propagate itself on an animal feed. Therefore, an intake rateof the feed for animals was sometimes lowered because of heat generationor bad smell of the feed caused by the propagation of the mold. Inaddition, a hopper opening of a feed tank sometimes clogged up by thepropagation of the mold thereby blocking the feed from discharging. Onthe other hand, an antibiotic substance having an antifungal activity oran antifungal agent such as a formic acid was sometimes added in theanimal feed in order to prevent and remove the propagation of the mold.However, there were a problem that the antifungal agent caused erosionand problems that the antibiotic substance caused the animals diarrheaor indigestion or caused emergence of drug-resistant bacteria orabnormal intestinal flora or the like to the animals. Conventionally,lactic acid bacteria having the antifungal activity are little knownexcept Lactobacillus sanfranciscencis (refer to Patent PublicationNo. 1) and Lactobacillus plantrum (refer to Non-Patent Publication No.1).

Conventionally, the lactic acid bacteria are also used as probiotics. Assuch ones, there are many proposals: a veterinary medication containinglactobacillus salivarius (refer to PATENT PUBLICATION 2), a deodorantfeed containing at least 0.1% of mixed microorganisms mainly compose ofmore than N×106 of lactic acid coccus and sporeformer (refer to PatentPublication No. 3), a feed additive adding an antioxidant substance andliving microbial agent to a high-class organic matter, putting them inan airtight container and fermenting them under a temperature of 25° C.(centirade) to 37° C. (centigrade) (refer to Patent Publication No. 4)and so on. “Probiotics” are also called a living microbial agent.According to the most common definition, it is a “living microbialadditive that works beneficially on a host animal by improving a balanceof an intestinal microbes” (Fuller, R.: Probiotics in man and animals.J. Appl. Bacteriol., 66, 365-378 (1989)). However, in these days, it mayalso be used in a broad sense as “microorganisms that beneficially workon health maintenance of a host” (Lee, Y. K. and Salminen, S.: Thecoming of age of probiotics, Trends Food Sci. Technol. 6, 241-245(1995)). Thus, there is also a thought that even killed bacteria areincluded in the probiotics (Salminen, S. et al.: probiotics: how shouldthey be defined?, Trends Food Sci. Technol., 10, 107-110 (1999)).Typical microorganisms as the probiotics are lactic acid bacteriaincluding bifidobacteria. In addition, bacillus subtilis, butyric acidbacteria, propionic acid bacteria, yeast and the like are also used asthe probiotics.

PATENT PUBLICATION NO. 1: Japanese Laid Open Patent Publication No.2002-291466

NON-PATENT PUBLICATION NO. 1: Paola Layermicocca et al. Applied andEnvironmental Microbiology, September 2000, p4084˜p4090, vol. 66, No 9)

PATENT PUBLICATION NO. 2: Japanese Laid Open Patent Publication No.S50-132115

PATENT PUBLICATION NO. 3: Japanese Laid Open Patent Publication No.H9-322714

PATENT PUBLICATION NO. 4: Japanese Laid Open Patent Publication No.2001-299230

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

However, there has been no report about Lactobacillus delbrueckii havingantifungal activity until now. There is a suggestion to use the lacticacid bacteria alone about the lactic acid bacteria used as conventionalprobiotics as described in the Patent Publication No. 2. Still, asufficient effect has not been acquired. Therefore, as mentioned above,the majority is occupied by the one using the lactic acid bacteria incombination with the other microorganisms or the one combining otheruseful materials. Lately there is little suggestions on the lactic acidbacteria capable of obtaining sufficient probiotics solo.

In view of the above-mentioned circumstances, an object of the presentinvention is to provide novel lactic acid bacteria that have antifungalactivity and that are excellent in probiotic activity even in case ofuse solo.

Means to Solve the Problems

In order to solve the above-mentioned problem, novel lactic acidbacteria according to a first aspect of the present invention has asubject matter in Lactobacillus delbrueckii having antifungal activity.

Lactobacillus delbrueckii ANTI MUFFA (FERM P-19705) is preferable as theLactobacillus delbrueckii.

Lactobacillus delbrueckii preferably consists of Lactobacillusdelbrueckii having antifungal activity against Penicillium mold.

An animal feed according to a second aspect of the present invention isadded with the aforementioned Lactobacillus delbrueckii.

An animal drink according to a third aspect of the present invention isadded with the aforementioned Lactobacillus delbrueckii.

Living lactic acid bacteria agent according to a fourth aspect of thepresent invention has the aforementioned Lactobacillus delbrueckii as anactive ingredient.

An animal feed additive according to a fifth aspect of the presentinvention has the aforementioned Lactobacillus delbrueckii as an activeingredient.

An improving method of animal breeding according to a sixth aspect ofthe present invention uses the aforementioned Lactobacillus delbrueckii.

An improvement of the animal breeding is preferably one of animprovement of deodorization of an excretory substance of animals, animprovement of a survival rate of animals, an improvement of anegg-laying rate of hens for egg collection and an improvement of milkquality of milk cows. However, the improvement of the animal breedingbroadly contains and includes effects acquired by the probiotic activitythat the lactic acid bacteria of the present invention have.

A fermented food according to a seventh aspect of the present inventionis manufactured by use of the aforementioned Lactobacillus delbrueckii.

A manufacturing method of a fermented food according to an eighth aspectof the present invention uses the aforementioned Lactobacillusdelbrueckii.

A preservation method of a fermented food according to a ninth aspect ofthe present invention uses the aforementioned Lactobacillus delbrueckii.

A cleaning method of a stable according to a tenth aspect of the presentinvention disperses the aforementioned Lactobacillus delbrueckii on afloor or a ground of the stable.

A cleaning method of a water according to an eleventh aspect of thepresent invention disperses the aforementioned Lactobacillus delbrueckiidirectly into the water or houses them in a permeable case so as toimmerse and deposit them in the water.

EFFECTS OF THE INVENTION

Since the novel lactic acid bacteria according to the present inventionhave the antifungal activity, the novel lactic acid bacteria are usefulfor controlling the mold during preservation of foods, controlling themold of animal feeds or the like. Consequently, it is possible to reduceproblems caused by adding antibiotic substances or antifungal agents.Moreover, since the novel lactic acid bacteria according to the presentinvention are excellent in the probiotic activity, the novel lactic acidbacteria improve the intestinal flora. Consequently, they improvebreeding of a host such as a domestic fowl or a domestic animal. Inaddition, the same effects are performed in the animal feed, the animaldrink, the living lactic acid bacteria agent, the animal feed additive,the improvement method of the animal breeding, the fermented food, themanufacturing method of the fermented food, the preservation method ofthe food, the cleaning method of the stable and the cleaning method ofthe water each using the above-mentioned novel lactic acid bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a phylogenetic system of Lactobacillus delbrueckii ANTIMUFFA (FERM P-19705) by 16S rDNA gene sequence analysis.

FIG. 2 is a picture image showing a livestock feed of a firstcomparative example.

FIG. 3 is a picture image showing a livestock feed of a first practicalexample.

FIG. 4 is a graph showing a temporal change of antifungal activity andpH in 10% skim milk of Lactobacillus delbrueckii ANTI MUFFA (FERMP-19705) according to the present invention while compared with those ofa comparative example (Examination Case No. 4).

FIG. 5 is a picture image showing antifungal activity in case of addingLactobacillus delbrueckii ANTI MUFFA (FERM P-19705) according to thepresent invention to a feed while compared with that of a comparativeexample (Examination Case No. 5).

FIG. 6 is a photomicrograph image showing contaminated green moldisolated from the feed of the comparative example in FIG. 5.

FIG. 7 is a picture image showing antifungal activity in case of addingLactobacillus delbrueckii ANTI MUFFA (FERM P-19705) according to thepresent invention to a feed while compared with that of a comparativeexample (Examination Case No. 6).

BEST MODE FOR CARRYING OUT THE INVENTION

Lactobacillus delbrueckii of the present invention is preferablyLactobacillus delbrueckii ANTI MUFFA, which may be referred to as“present bacteria” or “present strain” hereafter. This strain has beendeposited in National Institute of Advanced Industrial Science andTechnology, Patent Deposit Center (Central 6, 1-1-1, Tsukuba, IbarakiJapan), a trust number of which is FERM P-19705.

The present bacteria were discovered in searching antibacterial activityof lactic acid bacteria. As shown in FIG. 1, the present bacteria has99.5% homology to Lactobacillus delbrueckii subspecies Lactis(Lactobacillus delbrueckii subsp. Lactis) as a result of a lineageanalysis by 16s rDNA base sequence analysis. Thus, it is thought to be astrain that belongs to Lactobacillus delbrueckii subspecies Lactis(Lactobacillus delbrueckii subsp. Lactis). Hereunder shown aremycological characteristics of the Lactobacillus delbrueckii ANTI MUFFA(FERM P-19705) cultivated by use of a liquid culture medium for passageof lactic acid bacteria, which had a distilled water added to 5.0 g ofglucose, 5.0 g of lactose, 10.0 g of tryptone, 5.0 g of yeast extract,1.0 g of Tween 80 and 9.1 g of L-cysteine hydrochloride into a totalamount of 1000 ml and which was sterilized by a process of steamingunder pressure at 121° C. for 15 minutes after being adjusted to pH6.8to pH7.0. With respect to sugar assimilation, it was cultivated by useof a basal medium for sugar fermentability test, which had a distilledwater added to 10.0 g of tryptone, 5.0 g of yeast extract and 0.06 g ofbromcresol purple into a total amount of 1000 ml and which wassterilized by a process of steaming under pressure at 121° C.(centigrade) for 15 minutes after being adjusted to pH6.8.

Cell Shape: Bacillus, Spore Formation: Nil, Mobility: Nil, Gram Stain:+(plus), Behavior against Oxygen: Facultative Anaerobic, Growth at 15°C.: −(minus), Growth at 45° C. (centigrade): +(plus), LacticFermentation: Homo−(minus), Gas Generation from Glucose: −(minus),Ammonia Generation from Arginine: −(minus), Hydrolyzability of SodiumuHippurate: −(minus), Tolerance to 4% Sodium Chloride: −(minus), SugarAssimilation: Arabinose−(minus), Xylose−(minus), Rhamnose−(minus),Ribose−(minus), Glucose+(plus), Mannose+(plus), Fructose+(plus),Galactose+(plus), Sucrose+(plus), Maltose+(plus), Cellobiose+(plus),Lactose+(plus), Trehalose+(plus), Melibiose−(minus), Raffinose−(minus),Melezitose−(minus), Mannitol−(minus), Sorbitol−(minus), Esculin+(plus),Salicin+(plus), Amygdalin−(minus), Sodium Gluconate−(minus), ProducedLactic Acid: D-Lactic Acid, Stereoisomerism of Produced Lactic Acid(HPLC, 24 Hrs): D(−)(90.7%), L(−)(9.3%), DNA GC Content (HPLC): 48.7%

The present bacteria can be incubated by use of an MRS medium, a TYLGmedium, a milk medium or the like which is common to the incubation ofthe lactic acid bacteria. An incubation temperature is at 20° C.(centigrade) to 50° C. (centigrade), preferably at 30° C. (centigrade)to 40° C. (centigrade). An incubation pH is at 3.5 to 9.0, preferably atpH 4.5 to 7.0. An incubation time is preferably for 6 to 30 hours. Thepresent bacteria have antifungal activity, particularly, antifungalactivity against Penicillium mold. The present bacteria have a blockingfunction against Penicillium olsonii that easily breeds in an animalfeed. Moreover, the present bacteria have a blocking function againstPenicillium chrysogenum and Penicillium roquefortii having mycotoxin(fungal toxin) producibility. Furthermore, the present strain stronglycontrols or inhibits growth of Kluyveromyces marxianus variety lactis(Kluyveromyces marxianus var. lactis 5Y307).

The present bacteria are added for use in a livestock feed for domesticfowls, cattle, swine or the like, a pet feed for dogs, cats or the like,an aquaculture feed for fish such as eels or the like, so as to controlor inhibit the mold of the feed by the antifungal activity thereof.Moreover, the present bacteria may be added in a drink such as drinkingwater for domestic fowls, domestic animals or pets.

The present bacteria can be used for a living or viable lactic acidbacteria agent that has the present bacteria as an active ingredient.The viable lactic acid bacteria agent may be commonly prepared by thepresent bacteria alone. Alternatively, it may be mixed with a substanceas a nutrient source of the present bacteria, such as corn, bran, ricebran or the like. In addition, the living lactic acid bacteria may beused in combination with other microorganisms or other useful materialsthat may not block or disturb the growth of the present bacteria. Theliving lactic acid bacteria can be added for use in the animal feed orthe animal drink.

The present bacteria satisfy safety, so that it is applicable to astarter for fermented foods. Moreover, the present bacteria have theantifungal activity, so that it is added so as to control and preventpathogenic bacteria or putrefactive bacteria. Thus, the present bacteriacan be used for preservation of foods.

In addition, the present bacteria can be used as probiotics. Probioticsare defined as orally ingestible microorganisms that bring aboutbeneficial effects on a host by improving intestinal flora of the host.The beneficial effects brought about to the host means a variety ofeffects that are obtained by ingestion of animals: promotion of animalgrowth, improvement of a feed demand rate, prevention and betterment ofenteropathy, deodorization of excretory substances, improvement ofsurvival rate, increase in egg-laying rate of egg collection chickens,improvement of milk quality of milk cows or the like. It has the samemeaning as improvement of animal breeding. The present bacteria is ableto improve the animal breeding by the probiotic activity by making theanimals ingest the animal feed added with an animal feed additive havingthe present bacteria as the active ingredient.

FIRST PRACTICAL EXAMPLE

While the present invention is described in detail while showingpractical examples, the present invention is not limited to thefollowing practical examples.

MANUFACTURING EXAMPLE

The present bacteria were inoculated in a MRS medium and incubated at30° C. (centigrade) to 40° C. (centigrade) for 24 hours for primaryculture. Then, a culture fluid was sprayed on a sterilized bran andincubated at 30° C. (centigrade) to 40° C. (centigrade) for three daysfor secondary culture so as to obtain a living lactic acid bacteriaagent. A bacterial count of the present bacteria contained in theacquired living lactic acid bacteria agent was 5.3*109/g.

[Examination Case No. 1 of Antifungal Activity]

Mold spores of a test bacterial strain in Table 1 was smeared on askim-milk agar plate (10 ml of sterilized 10% skim-milk, 5 ml of 2% agardissolved with BCP (100 mg/l), 150 μl (micro liter) of TYLG culturefluid (containing 106/ml of the present bacteria)). Then, 5 ml of 0.15%soft agar added with 50 μl (micro liter) TYLG (containing the 106/ml ofthe present bacteria) was overlaid thereon. Thereafter, the bacteriawere cultured at 30° C. (centigrade) for 2 days. After the culture, ablocking function against propagation of the mold was assessed incomparison with a control having no mold spores smeared thereon. As aresult, as shown in Table 1, it showed a strong inhibiting functionagainst the mold of the genus Penicillium.

TABLE 1 Blocking Function of Kind of Mold Antifungus Aspergillus flavusvariety flavus − (minus) (Aspergillus flavus var. flavus, JCM10252)Aspergillus niger − (minus) (Aspergillus niger, JCM5546) Aspergillusoryzae variety oryzae − (minus) (Aspergillus oryzae var. oryzae,JCM2239) Fusariumu oxysporum f. species − (minus) cucumerinum (Fusariumuoxysporum f. sp. cucumerinum, JCM9284) Geotrichum candidum − (minus)(Geotrichum candidum, JCM1747) Penicillium candidum − (minus)(Penicillium candidum, SAM-2) Penicillium chrysogenum + (plus)(Penicillium chrysogenum, JCM2056) Penicillium olsonii + (plus)Penicillium roqueforti + (plus) (Penicillium roqueforti, PR-2) Rhyzopusoryzae − (minus) (Rhyzopus oryzae, JCM 5557)

[Examination Case NO. 2 of Antifungal Activity (First Practical Example]

A test was conducted whether it was possible to control and inhibitpropagation of mold on a livestock feed by adding the present bacteriato the livestock feed. One gram (5.3*109 in number) of the living lacticacid bacterial agent obtained in the aforementioned manufacturingexample was added to 20 gram of the animal feed (60 wt % of cereal(corn, rye), 26 wt % of oil-seed cake and meal, 2 wt % of chaff and branand 12 wt % of other materials (sweet stuff waste, mollasses or thelike)). Then, such animal feed was added with 20 ml of distilled waterand left out for 377 days at room temperature while housed in a cup(First Practical Example). 20 gram of a similar livestock feed was addedonly with 20 ml of distilled water and left out in the same manner(First Comparison Example). As a result, as shown in FIG. 2 and FIG. 3,Penicillium olsonii as contaminated mold of the livestock feedpropagated on the feed and the feed changed color in blue green in thefirst comparative example. In contrast, the Penicillium olsonii wascontrolled and prevented from propagation in the first practicalembodiment. The first comparative example in FIG. 2 shows a state after31 days from the neglect, while the first practical embodiment 1 in FIG.3 shows a state after 377 days from the neglect.

[Examination Case No. 3 of Antifungal Activity]

An examination was conducted on antifungal activity of the presentstrain that was screened by a milk agar plate method. A Penicilliumroqueforti PR-1 strain, which may be referred to as “PR-1 strain”hereafter, was used as an indicator mold. First, 10 ml of 10% skimmedmilk, 5 ml of 2% agar solution containing BPC (100 mg/l) and 150 μl ofthe culture fluid of the present strain were mixed. After the agarhardened, mold spores of the PR-1 strain that was diluted ten times wassmeared thereon. In preparing the mold spores, a potate dextrose agarslant medium cultured at 25° C. (centigrade) for 7 days was added withnormal saline solution containing Tween 80 (0.1%) and stirredthereafter. Then, there were provided for test spores prepared byfiltration through a glass wool.

Next, 5 ml of a soft agar (0.15%) added with 50 μl (micro liter) of aculture fluid of the present strain culture was overlaid thereon andincubated at 30° C. (centigrade) for 2 to 5 days. Consequently, it wasconfirmed that growth of the mold (PR-1 strain) was obviously restrainedin a culture medium (test plate) containing the present strain havingthe antifungal activity in comparison with a culture medium (controlplate) that cultivated the mold (PR-1 strain) solo. Thus, a degree ofgrowth of the mold (PR-1 strain) was compared between the control plateand the test plate, thereby measuring the antifungal activity of thepresent strain.

Next, an antifungal activity spectrum of the present strain against 10strains of the mold was measured. This measurement was conducted afterincubated the milk agar plate at 30° C. (centigrade) for 5 days.Consequently, it was confirmed that the present strain had theantifungal activity against three bacteria strains of genus Penicilliumthat had a bad influence on a feed, that is, Penicillium chrysogenum(JCM2056), Penicillium olsonii (wild type) and Penicillium roqueforti(PR-1). Among them, it was confirmed that the Penicillium chrysogenum(JCM2056) was perfectly controlled and restrained from growth.

[Examination Case No. 4 of Antifungal Activity]

Temporal changes of antifungal activity (Examination Case No. 4) and pHin a 10% skim milk of the present strain were examined by a colony platecount method. A line graph (three lines) in FIG. 4 shows pH of the skimmilk. A first line having triangular points shows the temporal change ofthe pH of the skim milk that inoculated only the present strain. Asecond line (uppermost line) having rhomboid points shows the temporalchange of the pH of the skim milk that inoculated only the Penicilliumroqueforti PR-1 strain. A third line (line generally overlapped with thefirst line) having circular points shows the temporal change of the pHof the skim milk that inoculated both the Penicillium roqueforti PR-1strain and the present strain. As shown in the line graph in FIG. 4, thepH goes down in the skim milk that inoculated only the present strainand in the skim milk that inoculated both the Penicillium roquefortiPR-1 strain and the present strain. However, there was not found anydecrease in the pH of the skim milk that inoculated only the Penicilliumroqueforti PR-1 strain.

A bar graph (three bars) in FIG. 4 shows a number of the bacteria in thepresent strain and the PR-1 strain. The bar graph at a left side showsthe number of the bacteria of the present strain that was incubatedsolo. The graph at a center shows the number of the bacteria in the PR-1strain that was incubated solo. The bar graph at a right side shows thenumber of the PR-1 strain when added with the present strain. As shownby the bar graph in FIG. 4, the PR-1 strain when added with the presentstrain was controlled and strained from propagation, and no residualbacterial count of the PR-1 strain was detected after 36 hours. Hence,it is obvious that the present strain is preferably applicable even to adairy product (fermented food) such as yoghurt or the like, for example.

[Examination Case No. 5 of Antifungal Activity]

A feed for egg-laying hens added with the present strain and a feed foregg-laying hens added with no present strain were left out for one year,respectively. Then, generation states of mold were checked. In FIG. 5, apicture image at a left side shows the generation state of the mold incase of adding no present strain in the feed for egg-laying hens. Apicture image of a right side in FIG. 5 shows the generation state ofthe mold in case of adding the present strain to the feed for egg-layinghens. As shown in FIG. 5, the feed for egg-laying hens added with nopresent strain gets moldy and totally decomposed. However, no moldgeneration is confirmed on the feed added with the present strain, andthe same feed is not decomposed. Moreover, as shown in FIG. 6, when thecontamination mold is separated for observation from the feed foregg-laying hens that had no present strain added and got rotted, aconidiophore or conidiospores lined up behind each other like foursterigmas. Hence, this mold was identified as the Penicillium olsoniifrom the results of its configuration, grown in a selection medium andtaxonomic characteristics. By such results, it was confirmed that thepresent strain had a strong control effect on the growth of thePenicillium olsonii.

[Examination Case No. 6 of Antifungal Activity]

Antifungal activity of the present strain against the Penicilliumroqueforti PR-1 was examined in the same manner as the examination caseNo. 3. Growth states of the PR-1 strain were observed in the test plate(culture medium incubating the PR-1 strain added with the presentstrain) and in the control plate (culture medium incubating the PR-1strain solo). The incubation was carried out at 30° C. (centigrade) for5 days. Consequently, as shown in an upper side picture image (A) inFIG. 7, many growths of the PR-1 strain were observed in the culturemedium containing no present strain. On the one hand, as shown a lowerside picture image (B) in FIG. 7, only the present strain could beobserved and no growth of the PR-1 strain could not be observed in theculture medium containing the present strain.

[Examination of Deodorizing Function of Excretory Substance of DomesticFowl and Domestic Animal]

Ammonia concentrations were measured in a pig house, a chicken coop anda cow barn, odor eliminating, respectively, so as to examine adeodorizing function of the present bacteria as follows.

[Example for Pig Feed (Second Practical Example)]

As a second comparative example, there was prepared a pig feed (60 wt %of cereal (corn, rye), 26 wt % of oil-seed cake and meal, 2 wt % ofchaff and bran and 12 wt % of other substances (sweet stuff waste,molasses, calcium phosphate or the like) added with 0.2 wt % of bacillusnatto. Then, the pig feed of the second comparative example was servedfor a fixed time period for ingestion to 2000 pigs in total that werebred in a breeding house for postweaning little pigs, a rearing houseand a mother pig house, respectively. Thereafter, the ammoniaconcentrations were measured in the pig houses after the same period hadpassed. The ammonia concentrations were measured by installing anammonia gas detector tube (manufactured by GASTEC CORPORATION, 3DL) at aheight of 1.5 m in the pig houses.

On the other hand, there was prepared a feed by adding the living lacticacid bacterial agent obtained by the aforementioned manufacturing methodto the aforesaid pig feed so that each pig could ingest 4 g (2.12*1010)per day as a second practical example. Then, the pig feed of the secondpractical example was served for ingestion to each of the aforementioned2000 pigs after the breeding period by the pig feed of the secondcomparative example had passed. Thereafter, the ammonia concentrationswere measured after 20 days and 40 days from after the breeding periodby the pig feed of the second comparative example had passed,respectively, in the same manner as the second comparative example. Inother words, the test of the second practical example was conductedafter the test of the second comparative example was finished. As aresult, as shown in Table 2, the second practical example shows asignificant deodorizing function in comparison with the secondcomparative example. In particular, ammonia odor was scarcely generatedafter 40 days.

TABLE 2 Ammonia concentration (ppm) 2nd comparative 2nd practicalexample example 20 days after 40 days after Postweaning 32.6 8.67 ND(not more little pig than detection breeding house limit) Rearing house27 8.11 2.5 Mother pig house 16.1 0.7 ND (not more than detection limit)

[Practical Example for Feed for Egg-Laying Hens (Third PracticalExample)

As a third comparative example, there was prepared a feed for egg-layinghens (59 wt % of corn, 23 wt % of oil-seed cake and meal, 3 wt % ofchaff and bran, 1 wt % of animal-based feed (fish meal) and 14 wt % ofother substances (calcium carbonate, animal oil and fat, corn steepliquor or the like) added with 2.0 wt % of a commercially availablelactic acid bacteria material (composed of lactic acid bacteria, yeast,filamentous bacteria or fungi or the like, made by A Corporation). Then,the feed for egg-laying hens of the third comparative example was servedfor a fixed time period for ingestion to 12000 egg-laying hens (ISABrown). Thereafter, the ammonia concentration was measured in the henhouse.

On the other hand, there was prepared a feed by adding 1.0 wt % of theliving lactic acid bacterial agent obtained by the aforementionedmanufacturing method to the feed for egg-laying hens similar to theaforementioned as a third practical example. Then, the feed foregg-laying hens of the third practical example was served for ingestionto each of the aforementioned 12000 hens (ISA Brown) after the breedingperiod by the feed for egg-laying hens of the third comparative examplehad passed. Thereafter, the ammonia concentration in the hen house wasmeasured. Results were shown in Table 3.

Moreover, the same feed for egg-laying hens as the third embodiment wasserved for a fixed time period for ingestion to the aforementioned 12000egg-laying hens (ISA Brown) after the breeding period by the feed foregg-laying hens of the third comparative example had passed. The ammoniaconcentrations in the hen house were measured after the test started,that is, after 5 day, 18 days, 25 days and 37 days after the livinglactic acid bacterial agent was added. Results were shown in Table 4.Here, in the third comparative example and the third practical example,the measurement of the ammonia concentrations were carried out by thesame measuring method and the measuring device as the aforementionedsecond practical example. Moreover, the test of the third practicalexample was conducted over 15 months after the test of the thirdcomparative example was conducted over 15 months and an additive-freefeed for egg-laying hens was then served for three months for ingestionof the egg-laying hens. Furthermore, in each of the tests of the thirdcomparative example and the third practical example, the breedingconditions were made the same in order to adjust comparison conditions.For example, a wing number per chicken coop (area per one chicken) and achicken coop environment (equipment or the like) were made the same. Onthe other hand, a time lag was set for the breeding period in the formof a prior period and a latter period as mentioned above. Table 3 showsan average value of results that were periodically measured between theexamination periods of each 15 months of the third comparative exampleand the third practical example. As a result, as shown in Table 3, thedeodorizing or odor eliminating function of the present bacteria wassuperior even in case of the domestic fowls. Moreover, as shown in Table4, the odor eliminating effect increased with time. Furthermore, bettervalues were acquired in case of breeding by the feed for egg-laying hensof the third practical embodiment in comparison with the thirdcomparative example, in any of a survival rate (87.39% to 52.07%,improvement of 35.32%) and an egg production (3,698,683 to 3,389,253,increase of 309,430).

TABLE 3 Ammonia concentration (ppm) 3rd comparison example 3rd practicalexample In chicken house 19.8 3.0

TABLE 4 Before adding Ammonia concentration (ppm) present 3rd practicalexample strain 5 days after 18 days after 25 days after 37 days after23.00 10.8 3.00 4.36 1.79

[Practical Example for Milk Cow Feed (Fourth Practical Example)]

As a fourth comparative example, there was prepared a milk cow feed (5kg of corn, 3 kg of barley, 1.5 to 2.0 kg of beet, 2 kg of cotton seeds,1.2 to 1.3 kg of defatted soybeans, 0.8 kg to 1 kg of soybeans, 8 to 10kg of hay and 150 to 2001 of water) added with a commercially availablelactic acid bacterial material (composed of lactic acid bacteria, yeast,koji mold or the like, made by B Corporation) so that each of the milkcows could ingest 200 g per day. Then, the milk cow feed of the fourthcomparative example was served for a fixed time period for ingestion to130 milk cows (Holstein). Thereafter, the ammonia concentration wasmeasured in a cow barn.

On the other hand, there was prepared a feed by adding the living lacticacid bacterial agent obtained by the aforementioned manufacturing methodto the milk cow feed similar to the aforesaid feed so that each milk cowcould ingest 40 g per day as a fourth practical example. Then, the milkcow feed of the fourth practical example was served for a fixed timeperiod for ingestion to each of the aforementioned 130 milk cows.Thereafter, the ammonia concentration was measured in the cow barn. Thetest of the fourth practical example was conducted after having each ofthe aforementioned 130 milk cows ingest an additive-free milk cow feedfor a fixed time period after finishing the test of the fourthcomparative example. The measurement of the ammonia concentrations werecarried out by the same measuring method and the measuring device as theaforementioned second practical example. As a result, as shown in Table5, the deodorizing effect was superior even in case of the milk cow.

TABLE 5 Ammonia concentration (ppm) 4th comparison example 4th practicalexample In cow barn 5.52 ND (not more than detection limit)

[Examination of Egg-Laying Hen's Survival Rate and Egg-Laying Rate]

In the test over each 15 months carried out in the examination of thedeodorizing effect of the above-mentioned third practical example, asurvival rate and an egg-laying rate were also examined together withthe test of the deodorizing function. As described above, the thirdcomparative example 3 had the domestic fowls ingest the domestic fowlfeed added with the commercially available lactic acid bacterialmaterial. On the other hand, as described above, the third practicalexample had the domestic fowls ingest the domestic fowl feed added withthe living lactic acid bacterial agent. As a result, as shown in Table6, the survival rate of the egg-laying hens that ingested the presentbacteria was very high. Moreover, as shown in Table 7, the egg-layinghens that ingested the present bacteria were also excellent in theegg-laying rate.

TABLE 6 Survival rate (%) 3rd practical example 87.39 3rd comparativeexample 52.07

TABLE 7 Total egg production (number) Egg-laying rate (%) 3rd practicalexample 3,698,683 100 3rd comparative 3,389,253 91.6 example

[Examination of Improving Function of Milk Quality (Fifth PracticalExample)]

Milk cows were bred by using a milk cow feed (Fifth Comparison Example)similar to the feed (Fourth Comparison Example) that added thecommercially available lactic acid bacterial material to the milk cowfeed in the examination of the deodorizing function of theaforementioned fourth practical example and a milk cow feed (FifthPractical Example) similar to the feed (Fourth Practical Example) thatadded the living lactic acid bacterial agent obtained by themanufacturing method to the milk cow feed, respectively. Then, the milkcows were milked 17 times at intervals of ten days. Thereby, examinationwas performed on a milk fat percentage, a milk protein percentage and anon-fat milk solid content percentage of the milk. In the fifthcomparative example, 130 milk cows (Holstein) were served for ingestionwith the milk cow feed added with the commercially available lactic acidbacterial material. Moreover, the test of the fifth practical examplewas conducted by having the milk cows ingest an additive-free milk cowfeed for a fixed time period after finishing the test of the fifthcomparative example and thereafter by having the same 130 milk cows(Holstein) ingest the milk cow feed (feed of the fifth practicalexample) added with the living lactic acid bacterial agent obtained bythe manufacturing method. The milk fat percentage, the dairy proteinpercentage and the non-fat milk solid of each milk milked from the milkcows were measured by use of a milk constituent analyzing device (MilkoScan FT120 manufactured by FUJIHIRA INDUSTRY CO., LTD.). Table 8 showsan average of 17 time measurement values which went through the test. Asa result, as shown in Table 8, the milk fat percentage, the milk proteinpercentage and the non-fat milk solid content percentage of the milkproduced from the milk cows that ingested the present bacteria were allhigh compared with the case of the fifth comparative example and itsmilk quality was improved.

TABLE 8 Non-fat Milk Milk fat Milk protein solid content percentage (%)percentage (%) percentage (%) 5th practical 4.01 3.41 9.00 example 5thcomparative 3.91 3.29 8.82 example

From the above results, it is thought that the present bacteria ishighly fixable in intestines of domestic fowls or livestock so as toimprove digestion and absorption thereby to have the probiotic activityeven when the present bacteria is used solo. Therefore, the presentinvention can be concretized as the probiotics (living microorganismagent) and exhibits the above-mentioned effects. Moreover, as describedabove, the present invention can be embodied not only into the animalfeed additive but also into the animal feed added with the presentstrain or the animal drink added with the present strain. In this case,the present invention performs the same effects as the above.Furthermore, as described above, the present strain has the effects onimprovement of the animal breeding such as the livestock or the domesticfowls. In addition, if the present strain is added to foods, it is alsoapplicable to a use for the preservation of foods or food microorganismcontrol (biopreservation). In this case, the present strain performssuch a proper effect as not seen conventionally by the property or thecharacteristic as mentioned above. Moreover, as described in theexamination case No. 4 of the aforementioned antifungal activity, thepresent strain can be added to fermented foods so as to be used formanufacturing of the fermented foods. Thus, the present strain performssuch a proper effect as not seen conventionally by the property or thecharacteristic as mentioned above. In addition, the animal feed or thelike added with the present strain may be dispersed intentionally orspread unintentionally on a ground, a floor, a drain ditch or the likeof a stable or the like at the time of feeding to the animals or thelike. Then, the present strain controls the microorganisms at suchdispersed places. Thereby, the present strain cleans up or deodorizesthe dispersed places by the above-mentioned property or characteristicso as to perform the proper effects that has not been seenconventionally. Alternatively, the animal feed or the like added withthe present strain may be dispersed directly in the water or be sunk anddeposited in the water while being housed in a permeable case such as anet-shaped object. Then, the present strain controls the microorganismsin the water. Thereby, the present strain purifies or disinfects thewater by the above-mentioned property or characteristic so as to performthe proper effects that has not been seen conventionally.

1. A nitrogen oxide sensor electrode, comprising: a solid solution of anitrate or nitrite of an alkali metal; and an oxide of a rare-earthelement.
 2. The nitrogen oxide sensor electrode as set forth in claim 1,wherein said oxide is Eu₂O₃, Y₂O₃, or Gd₂O₃.
 3. The nitrogen oxidesensor electrode as set forth in claim 1, wherein said nitrate ornitrite is a nitrite of an alkali metal.
 4. The nitrogen oxide sensorelectrode as set forth in claim 3, wherein said nitrite is KNO₂.
 5. Anitrogen oxide sensor, comprising: the nitrogen oxide sensor electrodeas set forth in claim 1; a solid electrolyte; and an oxide ionconductor, wherein: the nitrogen oxide sensor electrode and the oxideion conductor are provided in contact with a surface of the solidelectrolyte; and the solid electrolyte conducts magnesium ions, aluminumions, rare earth ions, zirconium ions, or hafnium ions.
 6. The nitrogenoxide sensor as set forth in claim 5, wherein the solid electrolyte issandwiched between the nitrogen oxide sensor electrode and the oxide ionconductor.
 7. The nitrogen oxide sensor as set forth in claim 5, whereinthe solid electrolyte has a Nasicon or β-iron sulfate crystal structure.8. The nitrogen oxide sensor as set forth in claim 7, wherein the solidelectrolyte is a complex of Mg_(1+X)Zr₄P₆O_(24+X) (0<X≦0.4) andZr₂O(PO₄)₂ or a solid solution, Mg_(1−2Y)(Zr_(1−Y)Nb_(Y))₄P₆O₂₄ (0≦Y<½),conducting magnesium ions.
 9. The nitrogen oxide sensor as set forth inclaim 7, wherein the solid electrolyte has a composition,(Al_(0.2)Zr_(0.8))_(20/19)Nb(PO₄)₃, conducting aluminum ions.
 10. Thenitrogen oxide sensor as set forth in claim 7, wherein the solidelectrolyte has a composition, R_(1/3)Zr₂(PO₄)₃, conducting rare earthions, where R is a rare earth atom.
 11. The nitrogen oxide sensor as setforth in claim 7, wherein the solid electrolyte has a composition,ZrNb(PO₄)₃, conducting zirconium ions.
 12. The nitrogen oxide sensor asset forth in claim 7, wherein the solid electrolyte has a composition,HfNb(PO₄)₃, conducting hafnium ions.
 13. The nitrogen oxide sensor asset forth in claim 5, wherein the oxide ion conductor is made of atleast one of the group consisting of fully stabilized zirconia, ceriumoxide, bismuth oxide, hafnium oxide, thorium oxide, and lanthanumgallate.
 14. The nitrogen oxide sensor as set forth in claim 5, whereinthe solid electrolyte is 0.1 mm to 1.5 mm thick.
 15. The nitrogen oxidesensor as set forth in claim 5, wherein the oxide ion conductor is 0.1mm to 1.5 mm thick.
 16. The nitrogen oxide sensor electrode as set forthin claim 2, wherein said nitrate or nitrite is a nitrite of an alkalimetal.
 17. A nitrogen oxide sensor, comprising: the nitrogen oxidesensor electrode as set forth in claim 2; a solid electrolyte; and anoxide ion conductor, wherein: the nitrogen oxide sensor electrode andthe oxide ion conductor are provided in contact with a surface of thesolid electrolyte; and the solid electrolyte conducts magnesium ions,aluminum ions, rare earth ions, zirconium ions, or hafnium ions.
 18. Anitrogen oxide sensor, comprising: the nitrogen oxide sensor electrodeas set forth in claim 3; a solid electrolyte; and an oxide ionconductor, wherein: the nitrogen oxide sensor electrode and the oxideion conductor are provided in contact with a surface of the solidelectrolyte; and the solid electrolyte conducts magnesium ions, aluminumions, rare earth ions, zirconium ions, or hafnium ions.
 19. A nitrogenoxide sensor, comprising: the nitrogen oxide sensor electrode as setforth in claim 4; a solid electrolyte; and an oxide ion conductor,wherein: the nitrogen oxide sensor electrode and the oxide ion conductorare provided in contact with a surface of the solid electrolyte; and thesolid electrolyte conducts magnesium ions, aluminum ions, rare earthions, zirconium ions, or hafnium ions.
 20. The nitrogen oxide sensor asset forth in claim 6, wherein the solid electrolyte has a Nasicon orβ-iron sulfate crystal structure.
 21. The nitrogen oxide sensor as setforth in claim 5, wherein the nitrogen oxide sensor electrode is of aboard shape and 0.1 mm to 1.5 mm thick.